Genomic Assessment of Potential Probiotic Lactiplantibacillus plantarum CRM56-2 Isolated from Fermented Tea Leaves

Lactiplantibacillus plantarum is a widely studied species known for its probiotic properties that can help alleviate serum cholesterol levels. Whole-genome sequencing provides genetic information on probiotic attributes, metabolic activities and safety assessment. This study investigates the probiotic properties of strain CRM56-2, isolated from Thai fermented tea leaves, using Whole-Genome Sequencing (WGS) to evaluate the safety, health-promoting genes and functional analysis. Strain CRM56-2 showed bile salt hydrolase (BSH) activity, assimilated cholesterol at a rate of 75.94%, tolerated acidic and bile environments and attached to Caco-2 cells. Based on ANIb (98.9%), ANIm (99.2%), and digital DNA–DNA hybridisation (98.3%), strain CRM56-2 was identified as L. plantarum. In silico analysis revealed that it was not pathogenic and contained no antibiotic-resistance genes or plasmids. L. plantarum CRM56-2 possessed genes linked to several probiotic properties and beneficial impacts. The genome of strain CRM56-2 suggested that L. plantarum CRM56-2 is non-hazardous, with potential probiotic characteristics and beneficial impacts, which could enhance its probiotic application. Consequently, L. plantarum CRM56-2 demonstrated excellent cholesterol-lowering activity and probiotic properties.


INTRODUCTION
Lactic acid bacteria (LAB) are commonly used as probiotics because of their desirable features, such as safety, and longevity in the gastrointestinal tract (GIT) (Ye et al. 2020).Lactobacillus plantarum is mostly versatile of the extensively studied species, and it is found in fermented meat and plant sources (Siezen et al. 2010).The ability of L. plantarum to survive gastric and bile and its ability to survive and propagate in the GIT (Le & Yang 2018;Zhang et al. 2020) make it a promising target for probiotic research.In addition, L. plantarum is receiving attention in pharmaceutical sciences due to its cholesterol-lowering properties (Arasu et al. 2016).Probiotics can lower serum cholesterol using prebiotics to synthesise short-chain fatty acids (SCFAs), which can inhibit hepatic cholesterol synthesis, decrease serum lipids, and directly assimilate cholesterol (Pereira & Gibson 2002).
Several L. plantarum strains show potential probiotic traits, including adaptable growing characteristics, stress endurance, potent GIT survival and physiological roles, such as cholesterol-lowering (van den Nieuwboer et al. 2016).However, safety assessment is crucial in food and health applications, considering the growing concerns regarding antibiotic resistance and virulence factors.Therefore, whole-genome sequencing (WGS) analysis enables precise identification while providing molecular information on probiotic attributes, putative metabolisms and safety evaluation, such as virulent genetic elements, antibiotic resistance genes (ARGs), as well as genetic factors associated with risky substances (Guinane et al. 2016;Li et al. 2017).With the publication of whole-genome sequences of several L. plantarum strains in the NCBI database, comprehensive knowledge of L. plantarum's functional properties and innovative applications becomes achievable.The genomic analysis must define a probiotic characteristic of an interesting strain.Selection criteria were bile salt hydrolase (BSH) activity, the highest cholesterol assimilation ability, and one of the significant probiotic species.Furthermore, genomic characterisation of L. plantarum strains isolated from the Thai-fermented leaves of Camellia sinensis needs investigation.
Accordingly, the present study endeavored to assess both the cholesterollowering effects and probiotic properties of strain CRM56-2.Additionally, WGS was employed to appraise the safety and probiotic-associated genes of this strain CRM56-2.

Isolation
Strain CRM56-2 was isolated from Camellia sinensis obtained from the Chiang Rai province in Thailand.The sample (0.5 g) was added in De Man, Rogosa and Sharpe (MRS) broth and incubated at 37°C for 48 h-72 h.A loopful of the culture was streaked on MRS agar plates containing 0.3% (w/v) CaCO 3 .A single colony surrounded by a clear zone was selected and purified on MRS agar plates.The pure culture was preserved in 10% skim milk at -80°C and lyophilised.

Phenotypic characteristics
The strain's cell shape, size, arrangement and colonial appearance were observed by growing cells on MRS agar plates for two days.Gram staining was performed following the procedure described by Cowan and Steel (1965).The activity of catalase, reduction of nitrate, gas formation, hydrolysis of arginine, aesculin production, as well as slime formation were determined using the methods previously reported by Tanasupawat et al. (2002).Growth under different pH (3.5-10.0),temperatures (10,15,30,37,40,42 and 45°C),and NaCl concentrations (1,3,5,6,6.5,7,7.5,and 8% w/v) were evaluated in MRS broth.Acid production from carbohydrates was determined following the methods reported by Tanasupawat et al. (2002).The lactic acid isomer was analysed using the enzymatic method described by Okada et al. (1978).

16S rRNA gene sequencing analysis
The initial species identification of strain CRM56-2 was carried out using the 16S rRNA gene.It was amplified following the protocol by Phuengjayaem et al. (2017) and sequenced using universal primers [27F (5ʼ-AGAGTTTGATCMTGGCTCAG-3ʼ) and 1492R (5ʼTACGGYTACCTTGT-TACGACTTʼ3)] as described by Lane (1991) on a sequencer (Macrogen, Korea).Similarities of the 16S rRNA gene of strain CRM56-2 to the database were enumerated using the Ezbiocloud webbased tools (Yoon et al. 2017).The data were deposited in the DNA Data Bank of Japan (DDBJ), Mishima, Japan.The DDBJ accession number of strain CRM56-2 was LC742934.

Genomic sequencing, assembly and features
The genomic DNA was extracted following the procedure outlined by Yamada and Komagata (1970).Subsequently, the preparation of the library and sequencing were carried out at the Faculty of Life Sciences, Tokyo University of Agriculture, utilising the Nextera DNA Flex Library Prep Kit and the Illumina MiSeq platform with MiSeq v3 reagent kit (600 cycles).The genomic quality was determined using FastQC web-based tool, and TrimGalore web-based tool was applied to remove adaptors and low-quality reads.Filtered Illumina reads were assembled using Unicycler (Galaxy Version 0.4.8.0), and CheckM was used to evaluate the genomic quality (Park et al. 2015).The JSpeciesWS online server tool (Richter & Rosselló-Móra 2009;Richter et al. 2016) and Genome-to-Genome Distance Calculator (GGDC 2.1) (Meier-Kolthoff et al. 2013) were used to examine the average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) data.TYGS web server (https://tygs.dsmz.de/)was used to construct the phylogenomic tree (Meier-Kolthoff & Göker 2019).Furthermore, the circular genomic map was generated using the Proksee Server (Stothard et al. 2019).

Cell suspension preparation
Strain CRM56-2 was cultured in MRS broth for 24 h at 30°C.Subsequently, the cellfree supernatant was removed by centrifugation at 14,000 rpm for 10 min at 4°C.The cells were washed with phosphate buffer (0.1 M, pH 7.2) and resuspended in phosphate buffer (0.1 M, pH 7.2) to obtain a cell suspension of 10 9 CFU/mL.

BSH activity
The Kingkaew et al. (2022) approach was utilised to assess BSH activity.Taurodeoxycholic acid sodium salt (TDCA) [0.5% (w/v)] and calcium chloride (CaCl 2 ) [0.037% (w/v)] were added to MRS agar medium.A CRM56-2 cell suspension was spotted onto the agar, and the plates underwent a 72 h anaerobic incubation period at 37°C.Halos or opaque white colonies with halos around them suggested BSH activity.The control was MRS agar containing no TDCA and CaCl 2 .

Assimilation of cholesterol
Using MRS broth supplemented with cholesterol-polyethylene glycol (PEG) 600 (Sigma, India) at a final concentration of 100 µg/mL, the capacity of strain CRM56-2 to absorb cholesterol was assessed.The suspension of strain CRM56-2 (1%, v/v) was added into the MRS broth containing cholesterol-PEG 600 and incubated at 37°C for 24 h under anaerobic conditions.Following the procedure of Tomaro-Duchesneau et al. (2014), the cholesterol was isolated, and the concentration of cholesterol was quantified using the technique described by Rudel and Morris (1973).The cholesterol concentration was compared to a reference curve prepared using a cholesterol stock solution.The ability to assimilate cholesterol was determined by calculating the percentage of assimilated cholesterol (%) at each incubation, as follows:

Acid and bile tolerance properties
The acid and bile tolerance properties of strain CRM56-2 were determined according to the method of Hyronimus et al. (2000).For acid tolerance, 2% of LAB suspension (10 9 CFU/mL) was inoculated into MRS broth with a pH of 3.0 and 6.5, and incubated anaerobically at 37°C for 180 min.The samples were collected at 0 min and 180 min of incubation time to enumerate viable cells.For bile salt tolerance, 2% of LAB suspension (10 9 CFU/mL) was inoculated into MRS broth with a pH of 8.0, containing bile salt (0.3% (w/v)), as well as without supplementation of bile salt, and incubated anaerobically at 37°C for 3 h.The samples were collected at 0 min and 180 min of incubation time to count the remaining viable cells.The results were described as colony-forming units per milliliter (CFU/mL).

The ability of adhesion to Caco-2 cells
The adhesion ability of strain CRM56-2 to adhere to the intestinal epithelium was evaluated following the method reported by Bustos et al. (2012).Briefly, Caco-2 cells were inoculated into 24-well tissue culture plates at a concentration of 5 × 10 5 cells/mL and incubated at 37°C with 5% CO2 for 15 days, with culture medium changes every 72 h.Overnight cultures of strain CRM56-2 in MRS broth were collected by centrifugation at 14,000 rpm for 10 min at 4°C and cleansed with phosphate buffer solution.Subsequently, the strain CRM56-2 cells (10 9 CFU/mL) were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and inoculated onto the Caco-2 cells in each well, followed by incubation at 37°C with 5% CO2 for 90 min.The cells were washed thrice with PBS and lysed with 0.05% (v/v) Triton-X solution.The released bacterial cells were serially diluted, spotted onto MRS agar, and incubated at 37°C for 2 days.The adhesive capability was expressed as the percentage of adhesive cells to Caco-2 cells to the total sum of bacteria (CFU/mL).Lacticaseibacillus rhamnosus GG was used as a positive control.

Strain Identification
Strain CRM56-2 is a Gram-positive, catalase-negative, facultatively anaerobic rod.It synthesises DL-lactic acid from D-glucose homofermentative and does not produce gas from glucose.The strain can grow at a temperature range of 15°C-45°C, a pH range of 2-9, as well as in the presence of NaCl (1%-6% (w/v)).Based on the phylogenomic tree (Fig. 1), strain CRM56-2 was grouped with various strains of L. plantarum.A dDDH score of 98.3% was observed between strain CRM56-2 and L. plantarum DSM ATCC 14917 T and/or 20174 T , which was the highest dDDH value among closely related species (Table 2).Furthermore, this strain exhibited the highest ANIb and ANIm values of 98.92% and 99.27%, respectively, to L. plantarum ATCC 14917 T (Table 2).The ANI and dDDH values, which were higher than the species boundary value (ANI > 95%-96%), confirmed that strain CRM56-2 was unequivocally identified as L. plantarum (Chun et al. 2018).Therefore, conventional tests and genomic investigations confirm that strain CRM56-2 belongs to L. plantarum.

Genomic Features of Strain CRM56-2
Based on Table 1, L. plantarum CRM56-2 (JAEMUU00000000) had a genome size of 3,373,611 bp with an N50 of 216,722, L50 of 5, and a genome coverage of 437×.The DNA G+C content of CRM56-2 was 44.3%.The genome sizes and G+C content fell within the 3 Mb-3.6Mb range and 44%-45%, respectively.These values are consistent with previous reports for this species (Martino et al. 2016;Surve et al. 2022).This finding represents the inaugural report of L. plantarum isolation from a fermented plant source in northern Thailand.PGAP annotation reported 3,270 genes, including 3,079 coding genes, 111 pseudogenes, 80 RNA genes, 71 tRNAs and 4 ncRNAs.The genomic statistics are shown in Table 3. Strain CRM56-2 lacked CRISPRs and Supplementary Material Fig. 1 illustrates its subsystems.Fig. 2 shows the circular genome of strain CRM56-2.

CONCEIVABLE GENE FACTORS IN PROTEASE ACTIVITIES, METABOLISM OF CARBOHYDRATES AND BENEFICIAL METABOLITES OF L. PLANTARUM CRM56-2
Enzymes-associated genes with conserved proteolytic and metabolic sugar systems could be found in strain CRM56-2, allowing for the performance of a functional genomic investigation.The genome of strain CRM56-2 encodes several proteases, such as peptidases (pep), proteinase (prt), and an oligopeptide ABC transport system (opp).Peptidase enzymes cleave various compounds, including asparagine, casein, cysteine, glutamate-derived peptides, leucine, methionine, proline and serine (De Jesus et al. 2022) (Refer Supplementary Material Table S1 for details).
Various enzymes related to the breakdown and utilisation of carbohydrates, such as chitobiose, fructose, galactose, glucose, mannose and sucrose, were identified in the genome of L. plantarum CRM56-2.These include 6-phosphobeta-glucosidase, glucokinase, phosphoglucomutase and phosphomannose isomerase.Moreover, genes involved in transporting cellobiose, fructose, glucose and mannose, mainly through the PTS system, were also discovered (De Jesus et al. 2022) (Refer Supplementary Material Table S2 for details).
In summary, the abundance and diversity of CAZymes genes in L. plantarum CRM56-2 suggest that this strain has a strong potential for immunomodulation and pathogen prevention as a probiotic.
Several genes encoding essential enzymes involved in the fermentation process were identified in L. plantarum CRM56-2, including acetate kinase, glyceraldehyde 3-phosphate dehydrogenase, glucose-6-phosphate isomerase, glucokinase, phosphoglycerate kinase, phosphoketolase, pyruvate kinase, pentose-5-phosphate 3-epimerase, lactic acid dehydrogenase and others (De Jesus et al. 2022).These enzymes play a critical role in the production of acetate or lactate (De Jesus et al. 2022).The study also investigated genes associated with vitamin production, such as dihydrofolate reductase (B9), riboflavin kinase (B2), and thiamine pyrophosphokinase (B1), as well as butyrate-associated genes (Supplementary Material Table S3 for details) (Botta et al. 2017).KEGG annotation revealed butanoate metabolism in L. plantarum CRM56-2, with genes implicated in butyric acid synthesis linked to the complimentary functions of a medium-chain thioesterase and FASII in CRM56-2.
These metabolic characteristics help the CRM56-2 strain ferment substances and produce useful metabolites such bioactive peptides, lactate, SCFA and vitamins.Key enzymes include acetate kinase, glyceraldehyde-3phosphate dehydrogenase, lactate dehydrogenase, phosphoketolase, peptidases, proteinases, pyruvate kinase, thiamine pyrophosphokinase and riboflavin kinase are associated with the Embden-Meyerhof (EMP) or phosphoketolase pathways and proteolysis in strain CRM56-2.Moreover, the study investigated butyric acid production-associated genes linked to the complementary activities of the FASII pathway and the medium-chain acyl-ACP thioesterase.Based on these findings, the presence of these metabolic-associated genes was similar to the other L. plantarum strains (Botta et al. 2017).These findings could have illustrated a previously unexplained metabolic pathway for the production of butyric acid in L. plantarum.Furthermore, these beneficial bioproducts are helpful in GIT inflammation.

Cholesterol-Lowering Activities
This study assessed the BSH activity BSH and cholesterol assimilation ability of strain CRM56-2 to evaluate its hypocholesterolemic effects (Table 4).

BSH and Cholesterol Assimilation
This study examined the BSH activity BSH and cholesterol assimilation ability of strain CRM56-2 to assess its hypocholesterolemic effects.The strain exhibited BSH activity, confirmed by the choloylglycine hydrolase (bsh) gene, and demonstrated the ability to assimilate cholesterol at 75.94%.These properties indicate the presence of cholesterol-lowering effects on the host, making BSH activity a desirable probiotic characteristic according to the FAO/WHO Guidelines for the Evaluation of Probiotics in Food (FAO/WHO 2002).However, deconjugated bile due to BSH activity may pose safety concerns, as it can negatively impact lipid digestion, disrupt intestinal environments, lead to gallstones, and be converted to carcinogens.Fortunately, CRM56-2 lacked genes to generate secondary bile salts, indicating no safety concerns related to hazardous secondary bile compounds.
The genome analysis of strain CRM56-2 showed cholesterol assimilationassociated genetic elements, including fba, ccpA and glgP.The capacity of the strain to absorb cholesterol is explained by the genes linked with cholesterol assimilation, which encode membrane-related proteins that can bind to the cholesterol molecule and further integrate it into the cell.LAB may absorb cholesterol from the GIT by attaching to the surface and utilising putative enzymatic activities, affecting the process of cholesterol absorption (Kingkaew et al. 2023).The cholesterollowering effects-associated genes are summarised in Table 5.In conclusion, strain CRM56-2's capacity to deconjugate bile salts and assimilate cholesterol makes it a promising probiotic candidate with potential hypocholesterolemic effects on the host.

Acid and bile tolerance and adhesion ability
For the assessment of acid tolerance, strain CRM56-2 was inoculated in MRS broth with a pH of 3.0 and incubated for 180 min.The viability of the strain was reduced from 2.4 × 10 7 to 5.8 × 10 6 CFU/mL.To evaluate bile tolerance, strain CRM56-2 was incubated in MRS broth supplemented with 0.3% bile salt at pH 8.0 for 3 h.The viability of the strain slightly increased from 2.7 × 10 6 to 7.7 × 10 6 CFU/mL (Table 3).Furthermore, the adhesion ability of strain CRM56-2 and L. rhamnosus GG was found to be approximately 0.40 ± 0.15% and 0.40 ± 0.05%, respectively.

Probiogenomic characteristics
The genome of L. plantarum CRM56-2 contains encoded genes associated with stress response in the GIT and adhesion ability, including ATP-dependent ClpX protease, chaperones (GroeL, DnaJ, DnaK), enolase, F 0 F 1 ATP system genes, Na + /H + antiporter NhaC, glycine/betaine ABC transporter permease, S-ribosylhomocysteine lyase, two-component sensor histidine kinase, ornithine decarboxylase, serine protease HtrA, and others, as shown in Table 6.Probiotic traits are strain-specific, motivating searching for new superior strains.L. plantarum CRM56-2 showed excellent tolerance to acid and bile salt conditions.Additionally, analysis of the strain's genome indicated that its proteinaceous compounds play a role in environmental and genetic processing information and metabolic function, suggesting the relevance of these genes in maintaining the biological function of strain CRM56-2 in specific contexts or hosts.The strain's core genome contained genes involved in stress response mechanisms, such as bile efflux and proton extrusions, metabolic response, heat shock/chaperones protein synthesis expression and transcriptional regulators.These genetic components may be crucial to the bacterial endurance of strain CRM56-2 in the human GIT.Probiotic activity requires adhesion to the intestinal mucosa epithelium, and microbial surface proteins have been linked to colonisation (Ye et al. 2020).Genes encoding cell-surface proteins, such as elongation factor Tu and lipoprotein signal peptide, were found in L. plantarum CRM56-2.The strain also contained a gene encoding LPXTG-specific sortase, relevant to strain adherence to surrounding epithelial tissue.Cell-surface proteins known as sortase-dependent proteins are crucial for adhesion (Alayande et al. 2020).The adhesion-associated genes discovered in strain CRM56-2 may thus improve strain stability and aid in successful colonisation.

Safety assessment (pathogenicity, antibiotic resistance genes and mobile genetic elements)
After a comprehensive genome analysis, the strain CRM56-2 was identified and predicted to be a non-pathogenic microorganism (Table 7).The genome lacked mobile genetic elements such as plasmids and ARGs (Tables 1 and 7).Additionally, six prophage regions, including two intact prophages ranging in size from 29.5 to 44 Kb, three incomplete prophages (5.4 to 23.3 Kb), and one questionable prophage (18.8 Kb), were integrated into the chromosome (Supplementary Material Table S5 for details).Although virulent genetic elements were present, including the capsular polysaccharide biosynthesis protein (cps4E) and exopolysaccharides biosynthesis proteins (cps4B and cps2B), the genome of the strain CRM56-2 contained no ARGs.It was identified as a non-human pathogen, indicating that the strain is safe and minimises the chance of spreading ARGs to the host's gut normal flora.Phages are frequently found in the genomes of Lactobacillus species (probiotic), but no virulence factors or genes associated with pathogenic qualities were found in these phage regions.Prophages may improve bacterial fitness in unfavourable environments (Pei et al. 2021).Capsular-and exo-polysaccharide genes associated with virulence elements were explored.Exopolysaccharides improve the capacity of bacteria to survive under oxidative and osmotic stress and contribute to its ability to adhere (Diale et al. 2021).Additionally, commercial strains like the accepted Generally Recognised as Safe (GRAS) L. plantarum 299V contained the hemolysin III gene (hlyIII), as well as a number of Lactobacillus strains.If no additional virulence genes have been found in the genome, strains with the hlyIII gene are often regarded as benign.Numerous studies have been conducted on hemolysin III and its harmlessness in lactobacilli.Therefore, this proteinaceous compound is not a serious concern (Surachat et al. 2017).Because they increase bacterial survival, these genes are advantageous to the bacterium and may be required when viable cells are needed.

CONCLUSION
In this study, L. plantarum CRM56-2 was isolated from Camellia sinensis and found to express BSH activity, which was indicated by the development of an opaque white colony.Additionally, CRM56-2 could withstand acidic and bile salt environments, metabolise cholesterol by over 70%, and possibly attach to Caco-2 cells.The genomic assessment of L. plantarum CRM56-2 highlighted its appeal as a promising probiotic.The strain was deemed harmless due to the absence of ARGs, plasmids, and virulent genetic features.Furthermore, the strain contains several genes involved in the of acid and bile salts, adhesive ability, and other beneficial impacts.Based on the in vitro and in silico investigations, it was concluded that strain CRM56-2 has health-promoting benefits and probiotic features, making it a promising probiotic.The genetic information of this strain supported its favorable traits.

No
obtained from PATRIC; b = Data obtained from CheckM; c = Data obtained from RAST web-based tool; D = Data obtained from DFAST annotation; E = Data obtained from PlasmidFinder; f = Data obtained from NCBI.

Figure 1 :
Figure 1:The phylogenomic tree was generated using whole genome sequencing data from strain CRM56-2 and closely related type strains.

Table 2 :
ANIb and ANIm (%) and the digital DNA-DNA hybridisation (dDDH) values between the draft genomes of the strain CRM56-2; L. plantarum DSM ATCC and/or 14917 T 20174 T ; L. argentorensis DSM 16365 T ; L. paraplantarum DSM 10667 T and L. pentosus DSM 20314 T .Notes: *Recommended formula (identities/HSP length), which is liberated of genome length and is thus prosperous against the use of draft genome.

Table 4 :
In vitro probiotic properties and cholesterol-lowering activities of strain CRM56-2.
Note: Data obtained from DFast annotation.

Table 6 :
Predicted proteins identified in the genome of L. plantarum CRM56-2 strain involved in acid and bile tolerance and adhesion/interaction.

Table 7 :
Pathogenicity prediction, prophage detection and antibiotic resistance genes (ARGs) analysis from PathogenFinder and ResFinder of CGE and PHASTER (Default program settings applied).